Regulated degradation of HMG CoA reductase requires conformational changes in sterol-sensing domain

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is the rate-limiting enzyme in cholesterol synthesis and target of cholesterol-lowering statin drugs. Accumulation of sterols in endoplasmic reticulum (ER) membranes accelerates degradation of HMGCR, slowing the synthesis of cholesterol. Degradation of HMGCR is inhibited by its binding to UBIAD1 (UbiA prenyltransferase domain-containing protein-1). This inhibition contributes to statin-induced accumulation of HMGCR, which limits their cholesterol-lowering effects. Here, we report cryo-electron microscopy structures of the HMGCR-UBIAD1 complex, which is maintained by interactions between transmembrane helix (TM) 7 of HMGCR and TMs 2–4 of UBIAD1. Disrupting this interface by mutagenesis prevents complex formation, enhancing HMGCR degradation. TMs 2–6 of HMGCR contain a 170-amino acid sterol sensing domain (SSD), which exists in two conformations—one of which is essential for degradation. Thus, our data supports a model that rearrangement of the TMs in the SSD permits recruitment of proteins that initate HMGCR degradation, a key reaction in the regulatory system that governs cholesterol synthesis.

3-Hydroxy-3-methylglutaryl coenzyme A reductase (HMGCR) is a polytopic, endoplasmic reticulum (ER)-localized glycoprotein that catalyzes a rate-limiting step in synthesis of cholesterol and essential nonsterol isoprenoids such as farnesyl pyrophosphate and geranylgeranyl pyrophosphate (GGpp) 1 . HMGCR is tightly controlled by a complex feedback regulatory system that allows cells to constantly synthesize nonsterol isoprenoids while avoiding toxic overproduction of cholesterol and other sterols [2][3][4][5] . Part of this feedback control involves accelerated ERAD (ER-associated degradation) of HMGCR 6,7 . This ERAD is initiated by the accumulation of sterols in ER membranes, which triggers binding of HMGCR to ER membrane proteins called Insigs 8,9 . Insig binding is mediated by the N-terminal membrane domain of HMGCR, which is both necessary and sufficient for ERAD and contains eight transmembrane helices (TMs) that precede a large cytosolic catalytic domain 10,11 . TMs 2-6 of HMGCR comprise what is known as the sterol-sensing domain (SSD); mutation of a tetrapeptide sequence (Y 75 IYF) in the SSD of HMGCR abolishes its binding to Insigs, preventing ubiquitination and ERAD 9 . Insig-associated ubiquitin ligases mediate ubiquitination of lysines-89 and −248 (K89 and K248), which are exposed to the cytosol and lie adjacent to TMs 3 and 7 of HMGCR, respectively 9,12-14 . Sterol-induced ubiquitination marks HMGCR for extraction across ER membranes, after which it becomes dislocated into the cytosol for proteasomal degradation 15 . The combination of K89R and K248R mutations prevent sterol-induced ubiquitination and ERAD of HMGCR in both cultured cells and tissues of knock-in mice 9,16 .
Whereas Insigs accelerate the ERAD of HMGCR, another protein called UBIAD1 (UbiA prenyltransferase domain-containing protein-1) binds to and stabilizes the enzyme 17,18 . UBIAD1 was discovered as the enzyme that transfers the geranylgeranyl group from GGpp to menadione producing the vitamin K 2 subtype menaquinone-4 (MK-4) 19,20 . We subsequently identified UBIAD1 as a GGpp sensor that binds to HMGCR and inhibits its ERAD when ER membranes are depleted of GGpp 17 . When GGpp accumulates within ER membranes, the isoprene binds to UBIAD1, causing it to dissociate from HMGCR and translocate to the medial-trans cisternae of the Golgi 17,21 . Importantly, GGpp-induced translocation of UBIAD1 from the ER-to-Golgi occurs in HMGCRdeficient cells 22 . Dissociation from UBIAD1 allows for the maximal ERAD of HMGCR (Fig. 1a). The physiologic significance of the UBIAD1-HMGCR interaction is confirmed by the observation that missense mutations in UBIAD1 cause Schnyder corneal dystrophy (SCD), an autosomal-dominant eye disease characterized by corneal opacification owing to the over-accumulation of cholesterol 23,24 . SCD-associated variants of UBIAD1 are sequestered in the ER and resist GGpp-induced dissociation from HMGCR (Fig. 1a). As a result, SCD-associated UBIAD1 inhibits ERAD of HMGCR, which leads to enhanced synthesis and accumulation of cholesterol in both cultured cells and tissues of mice 17,21,22,25 .
Competitive inhibitors of HMGCR called statins are prescribed to lower circulating levels of low density lipoprotein (LDL)-cholesterol and reduce the incidence of atherosclerotic cardiovascular disease (ACVD) 26,27 . However, the efficacy of statins is reduced because they disrupt feedback control of HMGCR owing to depletion of sterol and nonsterol isoprenoids (including GGpp). This depletion leads to the b UBIAD1 BRIL HMGCR accumulation of HMGCR in the liver that overcomes inhibitory effects of statins, allowing continued synthesis of cholesterol that limits lowering of plasma cholesterol [28][29][30][31][32] . Our previous studies indicated that inhibition of ERAD substantially contributes to statin-induced accumulation of HMGCR, which correlates with ER sequestration of UBIAD1 16,25 . However, the molecular basis through which sterols and GGpp accelerate ERAD of HMGCR and how UBIAD1 blocks the reaction is unknown. Here, we determined cryogenic electron microscopy (cryo-EM) structures of HMGCR bound to SCD-associated UBIAD1 (N102S). Structural and functional analysis provide key insights into mechanisms for UBIAD1-mediated protection of HMGCR from ERAD. These findings have important implications for development of agents that enhance statin efficacy and further reduce ACVD. Moreover, our studies reveal that the HMGCR SSD adopts a specific conformation required for sterol-accelerated ERAD, establishing the molecular basis through which the region mediates regulation of cholesterol synthesis.

Assembly of a complex between HMGCR and UBIAD1
We focused on the stabilizing interaction between the membrane domain of hamster HMGCR and UBIAD1. The hamster proteins share over 95% overall identity with their human counterparts (Supplementary Fig. 1). Expression plasmids were prepared that encode the FLAGtagged membrane domain of HMGCR harboring arginine substitutions for K89 and K248 (designated HMGCR TM ) and Strep-tagged UBIAD1 from which we deleted a flexible N-terminal region (amino acids 1-40).
Notably, UBIAD1 containing the N-terminal deletion continued to localize to the Golgi of GGpp-replete cells ( Supplementary Fig. 2), indicating the protein was normally folded. HMGCR TM and UBIAD1 N102S were co-expressed in HEK-293 GnTIcells and purified by anti-FLAG chromatography. Gel filtration shows that the HMGCR TM -UBIAD1 N102S complex migrated as a single peak ( Supplementary Fig. 3a); the presence of both proteins in the peak fraction was confirmed by immunoblot and mass spectrometry. Unfortunately, cryo-EM images of the HMGCR TM -UBIAD1 N102S complex displayed limited features and we failed to reconstitute a 3D model. Hydropathy plots predict that UBIAD1 is comprised of 9 TMs. To provide a fiducial marker for particle image alignment in cryo-EM structure determination, we inserted the soluble, thermostabilized apocytochrome b562RIL (BRIL) 34 in a cytosolic loop predicted to localize between TMs 8 and 9 of UBIAD1 (designated UBIAD1-BRIL N102S ) (Fig. 1b). The yield and biochemical stability of the HMGCR TM -UBIAD1-BRIL N102S was considerably enhanced compared to the HMGCR TM -UBI-AD1 N102S complex. HMGCR TM -UBIAD1-BRIL N102S was assembled into complex with anti-BRIL Fab (Fab BRIL ) 34 and an anti-Fab nanobody (Nb) 35 in amphipols ( Supplementary Fig. 3c, d), of which the structure was determined by cryo-EM at a resolution of 3.6-Å ( Supplementary Fig. 4). Densities of all 9 TMs of UBIAD1 and TMs 1-7 of HMGCR were resolved. However, the cryo-EM map of HMGCR part does not provide sufficient resolution to determine the atomic structure ( Supplementary Fig. 4c)  To address this problem, we generated and screened ∼1000 hybridoma clones for conformation-specific antibodies using HMGCR TM -UBIAD1 N102S complex as antigen. We identified one monoclonal antibody designated IgG-15B2 that bound native HMGCR TM -UBIAD1 N102S , and found that BRIL insertion did not interfere the epitope recognition. Fab 15B2 , a Fab fragment derived from IgG-15B2, was co-purified with HMGCR TM -UBIAD1-BRIL N102S upon gel filtration ( Supplementary  Fig. 3b, e). The structure of the HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 complex was determined by cryo-EM at a resolution of 3.3-Å ( Fig. 1c, g, Supplementary Table 1). HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 complex existed as either a monomeric or dimeric heterotrimer. We observed clear densities for all 9 TMs of UBIAD1 and TMs 1-7 of HMGCR in the monomeric HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 heterotrimer (designated UBIAD1-HMGCR complex 1) (Fig. 1c, d, Supplementary Figs. 5, 6). Interestingly, within the dimeric structure of the HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 heterotrimer, each HMGCR TM in the complex existed in a different conformational state. The structure of one state, designated UBIAD1-HMGCR complex 2a, was determined at a resolution similar to that of complex 1 (Fig. 1f, Supplementary Figs. 5, 7). The structure of the other state, designated UBIAD1-HMGCR complex 2b, was resolved at a lower resolution. We observed clear cryo-EM map for TM1-7 of HMGCR in complex 2b ( Supplementary Fig. 7a); however, maps corresponding to TMs 7-9 of UBIAD1, BRIL, and Fab 15B2 failed to be observed. The cryo-EM map shows that the HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 complex 2b was rotated 180°in detergent compared to complex 2a (Supplementary Fig. 7a). This rotation is likely an artifact of detergent solubilization and unlikely to be physiological.

Overall Structure of the UBIAD1-HMGCR complex
Cryo-EM maps revealed that the Fab 15B2 epitope encompasses the cytosolic interface of the HMGCR TM -UBIAD1-BRIL N102S complex and includes regions of both proteins (Fig. 1c, e, Supplementary Fig. 8). The possibility exists that Fab 15B2 modulates complex formation by altering the structure of HMGCR TM and/or UBIAD-BRIL N102S . Structural analysis of HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 complex indicates that Fab BRIL and Nb disrupted the dimer interface ( Supplementary Fig. 9a, b); thus, the HMGCR TM -UBIAD1-BRIL N102S -Fab BRIL -Nb complex was only observed in the monomeric state. Although the overall resolution of HMGCR TM -UBIAD1-BRIL N102S -Fab BRIL -Nb complex is lower than that of the HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 complex, structural analysis revealed the UBIAD1-HMGCR interface was identical in both complexes (Supplementary Figs. 4 and 9c).
The conformation of UBIAD1 is identical in Complexes 1 and 2a (Fig. 2a). The interface between UBIAD1 and HMGCR occupies an area of~1000 Å 2 ; the complex is maintained by several interactions between TMs 2 and 4 of UBIAD1 and TMs 5 and 7 of HMGCR (Figs. 1d and 2a). The main chain of V101, which localizes to TM2 of UBIAD1, makes hydrophilic contact with N250 in TM7 of HMGCR (Fig. 2b). TM4 of UBIAD1 makes additional hydrophobic contacts with HMGCR and two hydrophilic interactions are observed between E158 of UBIAD1-TM4 and S271 of HMGCR-TM7 and between K156 of UBIAD1-TM4 and the main chain in S185 of HMGCR-TM5 (Fig. 2c). A hydrophobic interface is formed by interaction of residues in TM5 and TM7 of HMGCR with residues in TM2, TM3, and TM4 of UBIAD1 (Fig. 2b, c).
We next compared the association of UBIAD1 N102S with HMGCR TM and variants of the protein harboring mutations in the HMGCR-UBIAD1 interface predicted to disrupt complex formation (V181R, V252R, V256R, and V267R in HMGCR) (Fig. 2b, c). Because the HMGCR-UBIAD1 complex cannot be assembled in vitro, we used coimmunoprecipitation of the proteins expressed in vivo to measure their association. HEK-293 GnTIcells transfected with expression plasmids encoding Strep-tagged UBIAD1 N102S and FLAG-tagged HMGCR TM or its variants were lysed and precipitated with anti-FLAG-  TM1   pre-TM1   TM2   TM2 TM2  TM3   TM5   TM5   TM9   TM8   TM7   Hx6-7  Hx6-7   TM9  TM8   TM7   TM3 pre-TM1 The electrostatic surface representation of the catalytic cavity is shown on the right. The entrance of the catalytic cavity is indicated by an arrow. The N102S mutation is shown in sticks. b Overall structure of the UbiA homolog from Aeropyrum pernix (ApUbiA) viewed from the side of the membrane. The electrostatic surface representation of the catalytic cavity is shown on the right. The entrance of the catalytic cavity is indicated by an arrow. The putative substrate GSPP is shown in sticks and the Mg 2+ ions are shown in green balls. c Structural comparison of UBIAD1 and ApUbiA. The movements of TM1 and Hx6-7 are indicated by arrows. The potential clash between UBIAD1-TM1 and GSPP is indicated by a dashed oval. The HMGCR binding site is indicated by a blue line. d Distribution of SCD-associated mutation in UBIAD1. The mutations that locate in the cap domain are colored in orange and the mutations that locate in the catalytic core are colored in purple. coupled agarose beads. Immunoblot analysis of precipitated material revealed that as expected, UBIAD1 N102S co-precipitated with HMGCR TM (Fig. 2d, lane 3). In contrast, UBIAD1 N102S failed to be co-precipitated with HMGCR TM (V252R) and HMGCR TM (V256R) (lanes 5 and 6); HMGCR TM (V181R) and HMGCR TM (V267R) precipitated UBIAD1 N102S albeit at slightly reduced levels compared to HMGCR TM (lanes 4 and 7). We also generated a variant of UBIAD1 N102S containing a point mutation (F172A) at the HMGCR-UBIAD1 complex interface (Fig. 2b, c). The results show that co-precipitation of UBIAD1 N102S (F172A) with HMGCR TM was reduced compared to UBIAD1 N102S (compare lane 8 with lanes 3 and 9).
These motifs, which correspond to N 102 TYYDFSKG and D 236 MESD in UBIAD1, are located at the C-terminal ends of TMs 2 and 6, respectively (Fig. 3a). Hx6-7, L2-3, and the loop between TMs 4 and 5 (L4-5) form a cap domain that lies over a central, negatively charged cavity generated by TMs 1, 2, 4, 5, and 6 ( Fig. 3a). The structural analysis of the archaeal UbiA prenyltransferases suggests a model in which the cap domain adopts an open conformation in the absence of the isoprenyl substrate 37,38 . Binding of the substrate induces conformational changes in L2-3 that causes the cap domain to adopt a closed conformation that seals the enzyme's active site (Fig. 3b). The structure of UBIAD1 N102S reveals that the cap domain adopts an open conformation, which is consistent with previous findings that the N102S mutation reduces the affinity of UBIAD1 for GGpp 20 . Figure 3d shows the location of residues in UBIAD1 that are mutated in SCD. SCD-associated mutations cluster in L2-3 and Hx6-7 or line the central cavity that harbors the enzyme's active site. These residues, many of which are conserved in UbiA prenyltransferases, are likely involved in binding of GGpp or catalysis. Indeed, our group and others have demonstrated that introduction of SCD-associated mutations in UBIAD1 reduces enzymatic activity 20,33 .
In comparing the structures of archaeal UbiA prenyltransferases (bound to substrate) and UBIAD1 N102S , we noticed a significant difference. The central cavity in the archaeal enzymes has a lateral opening delineated by kinked TM1 and TM9 38 (Fig. 3b, c). This lateral opening may allow these enzymes to accommodate longer isoprenyl substrates and/or release reaction products. Interestingly, TM1 of UBIAD1 N102S forms an intact α-helix that blocks the lateral opening (Fig. 3a, c). It is tempting to speculate that binding to GGpp or MK-4 triggers conformational changes in TM1 that allows release of the product into the membrane bilayer.

Structural analysis of HMGCR TM
The overall structure of HMGCR TM revealed that TMs1-7 are integrated into membranes. The loops between TM1 and TM2 (25 amino acids in length) as well as TM8 were not visualized in the structure, which indicates considerable flexibility within the regions (Fig. 1d). Further analysis of the resolved structure revealed that HMGCR TM adopts two distinct conformations designated Conformation A and Conformation B (Fig. 4a, b). In UBIAD1-HMGCR complex 1 and 2b, HMGCR TM assumes Conformation A in which TM2 is perpendicular to the membrane, whereas TM4 is unwound to generate two half helices that we designate TM4a and TM4b (Fig. 4b). HMGCR TM assumes Conformation B in UBIAD1-HMGCR complex 2a. TM2 is tilted 45°in the membrane (Fig. 4a) and TM4 forms an intact α-helix (Fig. 4b). In the dimeric state of complex 2a, Conformation B becomes stabilized through direct interactions between TMs of HMGCR.
SSDs are found in five other proteins-Scap, Niemann-Pick C1 (NPC1), NPC1-Like1 (NPC1L1), Patched, and Dispatched-implicated in the regulation of cholesterol metabolism and signaling (Supplementary Fig. 10). Scap is a cholesterol-regulated escort protein required for activation of membrane-bound transcription factors called sterol regulatory element-binding proteins (SREBPs) 40 . NPC1 and NPC1L1 mediate intracellular transport of LDL-derived or dietary cholesterol 41 . Patched binds to the cholesterol-modified morphogen Hedgehog, while Dispatched mediates release of Hedgehog from cells 42 . Scap and HMGCR are unique among SSD-containing proteins in that sterols cause both proteins to bind Insigs. However, Insig binding does not lead to accelerated ERAD of Scap. Instead, the reaction traps Scap in the ER, preventing its transport to the Golgi for proteolytic activation of bound SREBPs 43 .

Dynamic Reorganization of TMs in the HMGCR SSD
Further analysis of the two HMGCR SSD conformations may provide insight into mechanisms for the sterol-sensing reaction. Thus, we superimposed the structure of TMs 1-6 of HMGCR in Conformation A  TM2   I132   I132   F80   R110   R110   TM1   TM1   TM3   TM3   TM5   TM6   TM5   TM6   TM3   TM3   F80   F80   TM2   TM3   d   R110   TM3   R110  I132   TM4   K102   D133   TM2   TM2   R110   TM3  TM3   I132   with the previously reported structure of the Scap-Insig-2 complex 50 . This superimposition indicates that the predicted HMGCR-Insig-2 interface is similar to the Scap-Insig interface, which is comprised of TMs 2, 4, and 5 of Scap and TMs 3 and 4 of Insig-2 (Fig. 5a). A previous finding suggested that unwinding of Scap-TM4 exposes negatively charged E359, permitting interaction with R110 (and perhaps K102) of Insig-2 that stabilizes the Scap-Insig-2 complex 50 . Our modeling predicts that D133 of HMGCR assumes a position equivalent to that of E359 in the broken TM4 of Scap and contributes to formation of the HMGCR-Insig-2 complex (Fig. 5b). The YIYF motif of Scap and HMGCR, which is required for their sterol-induced binding to Insigs 50 , is positioned similarly in TM2 of both proteins (Fig. 5b). Figure 5c shows the superimposed structures of the Scap-Insig-2 complex and HMGCR (TMs 1-6) in Conformation B that results in rotation of TM2 and TM4 approximately 180° (Fig. 5a, c). This rotation causes significant steric hinderance between F80 and Insig-2-TM3; steric clash is also observed between I132 in HMGCR and R110 of Insig-2 (Fig. 5c, d). It is notable that in the absence of Insig-2, Scap-TM2 is not observed in the cryo-EM maps, which indicates considerable flexibility. TM4 is not broken and may adopt a continuous α-helix similar to that of HMGCR-TM4 in Conformation B ( Supplementary Fig. 12) 49 .

Discussion
Previous studies have described an intricate pathway through which distinct lipids-sterols and GGpp-accelerate Insig-mediated ERAD of HMGCR 3 . A key breakthrough in the understanding of HMGCR ERAD came with the discovery that UBIAD1 binds to HMGCR and inhibits its ERAD. In the current studies, we analyze the structure of the HMGCR-UBIAD1 complex, which reveals a hydrophobic interface that is mediated by multiple interactions between the TMs of UBIAD1 and HMGCR (Fig. 1c-g). Mutation of key residues in this interface disrupts formation of the HMGCR-UBIAD1 complex and blunts UBIAD1-mediated stabilization of HMGCR (Fig. 2d-f). Identification of the HMGCR-UBIAD1 interface has important implications for the molecular basis of statin-induced accumulation of HMGCR that was described more than 40 years ago 2 . Studies in genetically-manipulated mice revealed that inhibition of ERAD substantially contributes to statin-accumulation of HMGCR 16 . UBIAD1 is sequestered in the ER of hepatic membranes isolated from statin-fed mice owing to depletion of GGpp 25 and unequivocal genetic evidence has been obtained that UBIAD1 is an inhibitor of HMGCR ERAD 36 . Taken together with the current study, we predict molecules that disrupt the HMGCR-UBIAD1 interface or mimic GGpp in stimulating ER-to-Golgi translocation of UBIAD1 will enhance ERAD of HMGCR, preventing its accumulation associated with statin therapy.
We captured the SSD of HMGCR in two distinct conformations, indicating TMs in the region undergo dynamic reorganization within ER membranes (Fig. 5e). Importantly, the cytosolicallyexposed sites for sterol-induced ubiquitination (K89 and K248) are identical in both conformations, ruling out the possibility that reorganization of the SSD alters access of the sites to ubiquitination machinery. We attenuated the structural rearragnment of TMs in the SSD through protein engineering and discovered that confining HMGCR to Conformation B (Fig. 5f) abolished its Insigmediated ERAD stimulated by 25-HC (Fig. 5g). Certain oxysterols such as 25-HC are known to bind Insigs and it has been postulated that oxysterol-bound Insig in turn, associates with the SSDs of Scap and HMGCR 40 . Taking our current study into consideration, it is reasonable to speculate that oxysterol-bound Insig only engages the SSD when it assumes Conformation A. It should be also noted that the cholesterol synthesis intermediate 24,25-dihydrolanosterol (DHL) accelerates Insig-mediated ERAD of HMGCR 3 , but does not bind to Scap or Insig. We hypothesize that the SSD of HMGCR directly senses the concentration of DHL embedded in ER membranes, causing the protein to change its conformation to engage Insigs for ubiquitination and ERAD to control the synthesis of cholesterol. Although the structures of other SSD-containing proteins have been determined [44][45][46][47][48][49][50][51][52][53] , these studies neither observed the dynamic reorganization of the SSD nor determined whether multiple conformations of the SSD modulated the protein's activity. Our findings provide structural evidence validated by functional assays that are beginning to disclose the molecular basis through which SSDs control protein function.
Despite the advance in the understanding of the HMGCR ERAD pathway, several questions remain outstanding. For example, molecular mechanisms underlying transition of the HMGCR-SSD between Conformations A and B regulated by DHL and oxysterols remain to be determined. The precise mechanism through which UBIAD1 inhibits HMGCR ERAD at a post-ubiquitination step in the reaction has not been elucidated. Finally, mechanisms whereby GGpp dissociates the HMGCR-UBIAD1 complex and stimulates ER-to-Golgi transport of UBIAD1 is unknown. Attempts to address these important questions utilizing a variety of approaches are currently underway.

Expression plasmids
A cDNA encoding the transmembrane domain (amino acids 1-356) of hamster HMGCR harboring mutations (K89R, K248R) that abolish the enzyme's sterol-induced ubiquitination was cloned into the pEZT-BM vector 54 with a N-terminal FLAG tag. The resulting expression plasmid is designated pEZT-BM-FLAG-HMGCR TM . The cDNA for hamster UBIAD1 containing a 40-amino acid deletion at N-terminus and the SCD-associated N102S mutation was cloned into pEZT-BM vector preceded by a StrepII tag. BRIL insertion was screened through different replacement for loops between TMs of UBIAD1. The final construct used for cryo-EM structure determination is the one with BRIL insertion located between Phe300 and Pro309. This expression plasmid is designated pEZT-BM-StrepII-UBIAD1-BRIL N102S . The following expression plasmids were described in the indicated reference: pCMV-HMGCR (TM1-8)-T7 encoding amino acids 1-346 of hamster HMGCR with 3 copies of the T7 epitope at the C-terminus under transcriptional control of the cytomegalovirus (CMV) promoter 8 ; pCMV-Myc-UBIAD1 (N102S) encoding human UBIAD1 containing a Myc tag at the N-terminus under control of the CMV promoter 17 ; pCMV-Insig-1-Myc, which encodes human Insig followed by six copies of the Myc epitope under control of the CMV promoter 43 ; and pCMV-HMGCR-7, encoding full-length hamster HMGCR followed by three copies of the T7 epitope under control of the CMV promoter 9 . Sitedirected mutagenesis of pCMV-HMGCR (TM1-8)-T7, pEZT-BM-FLAG-HMGCR TM , and pEZT-BM-StrepII-UBIAD1 N102S (without BRIL insertion) was carried out by two-step overlapping PCR. All mutations were verified by sequencing.

Expression and purification of anti-BRIL Fab and its nanobody
The anti-BRIL Fab (Fab BRIL ) was expressed in E. coli and purified as described 34 . The DNA sequence encoding the anti-Fab BRIL nanobody (Nb) was derived from PDB: 6WW2 55 and synthesized from Integrated DNA Technologies and cloned into pET-26b vector with a C-terminal 6×His tag. The nanobody was expressed in E. coli BL21-Gold (DE3) cells with 0.5 mM IPTG at 25°C overnight and purified by Ni-NTA chromatography and size-exclusion chromatography on a Superdex 200 column. The proteins were concentrated and frozen at −80°C for further use.

Generation of Fab 15B2
Monoclonal antibody (designated 15B2) of isotype IgG1 were raised in mice (NZBWF1/J, The Jackson Laboratory) at the Department of Molecular Genetics of the University of Texas Southwestern Medical Center. The antigen immunize mice was the HMGCR TM -UBIAD1 N102S complex that was reconstituted into Amphipol A8-35. Hybridomas were created by fusion of splenic B lymphocytes from hyperimmune mice to SP2-mIL6 mouse myeloma cells (ATCC, CRL-2016). We used the combined techniques of ELISA, immunoblot analysis, and immunoprecipitation to identify antibodies that preferentially bound to HMGCR TMs -UBIAD1 N102S complex in its native, but not SDS-denatured state. These efforts yielded a mouse monoclonal antibody designated IgG-15B2. To clone IgG-15B2, total RNA was isolated from the hybridoma by RNA extraction kit (Qiagen) following the manufacturer's protocol. Total RNA was subjected to reverse transcription reactions using Superscript III reverse transcription kit (Invitrogen) and the resultant cDNA was used as a template in PCR reactions with degenerate primers to amplify the variable regions. Sequences of the resulting PCR products were analyzed with the IMGT database (http:// www.imgt.org/) to determine the variable regions of the light chain and heavy chain, which were then cloned into shuttle vectors for light chain and the Fab region of heavy chain with a C-terminal 6×His tag, respectively 56 . The resulting constructs were co-transfected to HEK-293S GnTI − cells (ATCC) for expression at 37°C. After 72 h, the medium was harvest and applied to Ni-NTA gravity columns. Following several washes with buffer A containing 20 mM imidazole, bound material was eluted with buffer A containing 250 mM imidazole. The eluate was then applied to a Superdex-200 Increase size-exclusion chromatography column (GE Healthcare) in buffer A and peak fractions containing Fab 15B2 were collected for complex assembly.

Cryo-EM imaging and data processing
The freshly purified complexes samples were added to Glow discharged Quantifoil R1.2/1.3 400 mesh Au holey carbon grids (Quantifoil), blotted using a Vitrobot Mark IV (FEI), and frozen in liquid ethane. The grids were imaged in a 300 kV Titan Krios (FEI) with a Gatan K3 Summit direct electron detector (Gatan). Data were collected using SerialEM 57 at 0.83 Å/pixel or 0.842 Å/pixel. Images were recorded for 5-second exposures in 50 subframes with a total dose of~60 electrons per Å 2 . Data were collected in super-resolution mode and the parameters of data collection are summarized in the Supplementary Table 1.
For all the three samples, Dark subtracted movie stacks were normalized by gain reference and the motion correction was performed using MotionCor2 58 . The contrast transfer function (CTF) was estimated using CTFFIND4 59 . After particle picking by crYOLO 60 , the low-quality images and false-positive particles were removed manually. For the HMGCR TM -UBIAD1-BRIL N102S -Fab BRIL -Nb complex, three data sets were collected. After 2D-classification of data set 1 in CryoSPARC 61 , classes with clean background were selected to generate initial models for the 3D-classification. Map from the best 3D class showing clear features of micelle, Fab and Nb were used as a model to fish out "good" particles from all the three data sets via 3Dclassification. The resulting particles were subjected to the secondary 3D-classification with a mask in RELION-3 62 . Particles from the good classes were polished and 3D classified in RELION-3. The best class was selected for the final 3D-refinement in RELION-3. For HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 complexes, good classes from the initial 2Dclassification were select to generated initial models for the 3Dclassification of the entire particle set in CryoSPARC. The best 3D class was 2D classified and the classes showing features of monomer or dimer were selected separately to refine the best 3D model, respectively. The resulting maps were used as models for the further 3Dclassification. 3D classes containing monomeric or dimeric particles were subjected to 2D-classification in CryoSPARC to remove the heterogeneous particles. The remaining particles in the two classes were polished in RELION-3, followed by CTF refinement and final 3D refinement in CryoSPARC, respectively. For HMGCR TM (Δ40-55)-UBIAD1-BRIL N102S -Fab 15B2 complexes, monomeric and dimeric maps from HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 complexes were used as models for 3D-classification in CryoSPARC. Particles from the monomeric and dimeric classes were applied to 2D-classification and a secondary 3D-classification to further exclude the bad particles. The remaining particles in the two classes were CTF refined follow by the final local 3D refinement in CryoSPARC, respectively.

Model Construction, Refinement and Validation
For the HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 complex, a HMGCR TM -UBIAD1-BRIL N102S structure predicted by AlphaFold 63 and a Fab 15B2 structure predicted by Swiss-Model 64 were docked into the Complex-1 map as the initial model. The refined Complex-1 structure was docked into the Complex-2a region of the Complex-2 map used as the initial model for Complex-2a. Both structure models were manually built by COOT 65 , followed by refinement in real space using PHENIX 66 and in reciprocal space using Refmac with secondary-structure restraints and stereochemical restraints 67 . For cross-validation, the final model was refined with the half map 1 from the final 3D-refinement. The resulting model was used to calculate the model vs. map FSC curve against half map 1 and 2, respectively, using the Comprehensive validation module in PHENIX. MolProbity 68 was used to validate the geometries of the model. Structure figures were generated using PyMOL (http://www. pymol.org), Chimera 69 and ChimeraX 70 . The Complex-1 structure with the deletion of TMs 7-8 of UBIAD1 was docked into the Complex-2b region of the Complex-2 map as the initial model for Complex-2b. the Complex-2b structure was refined once in real space using PHENIX for figures. For the HMGCR TM -UBIAD1-BRIL N102S -Fab BRIL -Nb complex, HMGCR TM -UBIAD1-BRIL N102S structure from Complex-1 and Fab BRIL -Nb structure from PDB: 6WW2 were docked into map as the initial model. The structure model was manually built by COOT and refined once in real space using PHENIX for figure preparation. For HMGCR TM (Δ40-55)-UBIAD1-BRIL N102S -Fab 15B2 complexes, structure of HMGCR TM -UBIAD1-BRIL N102S -Fab 15B2 Complex-2a was docked into the monomeric and dimeric maps, respectively, as initial models. The structure models were manually built by COOT and refined once in real space using PHENIX for figure preparation.

Statistics and reproducibility
The experiments in Figs. 2d-f, 5g, h and Supplementary Figs. 2, 3a-c were repeated at least two times on different days. Similar results were obtained.

Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.